Calcium oxalate crystals trigger epithelial-mesenchymal transition and carcinogenic features in renal cells: a crossroad between kidney stone disease and renal cancer

Increasing evidence of association between kidney stone disease (KSD) and renal cell carcinoma (RCC) has been reported. Nevertheless, mechanism underlying such association remained unknown. Herein, we investigated the effects of calcium oxalate monohydrate (COM), a major crystalline component causing KSD, on induction of carcinogenic features in non-cancerous renal cells. COM crystals induced morphological changes from epithelial to fibroblast-like spindle shape. Additionally, COM increased spindle index and mesenchymal markers (fibronectin and vimentin) but declined epithelial markers (E-cadherin and zonula occludens-1). Moreover, COM down-regulated ARID1A, a tumor suppressor gene recently reported to be reversely associated with RCC, at both mRNA and protein levels. COM also down-regulated other RCC-related tumor suppressor genes, PTEN and VHL, but up-regulated oncogene TPX2. Finally, COM enhanced invading capability, cell-aggregate formation, chemoresistance to cisplatin, and secretion of an angiogenic factor (VEGF). These data indicate that COM crystals trigger epithelial-mesenchymal transition (EMT) and several carcinogenic features in the non-cancerous renal cells. These mechanisms may explain and strengthen the association between KSD and RCC. Supplementary Information The online version contains supplementary material available at 10.1186/s40164-022-00320-y.

added, and the stained cells were analyzed by using BD Accuri C6 flow cytometer (BD Biosciences). Three technical replicates were done per each biological sample.

Immunofluorescence cellular staining
The cells were maintained and treated as aforementioned but on coverslip in each well. After 24-h incubation with or without COM crystals, the cells were subjected to immunofluorescence stainings as previously reported [9,10]. Briefly, the cells were fixed with 4% paraformaldehyde at 25°C for 15 min followed by permeabilization with 0.1% Triton X-100 in PBS at 25°C for 15 min. Non-specific bindings were blocked with 1% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS at 25°C for 30 min. The cells were then incubated with rat monoclonal anti-E-cadherin (Santa Cruz Biotechnology; Santa Cruz, CA,), mouse monoclonal anti-fibronectin (Santa Cruz Biotechnology), mouse monoclonal anti-vimentin (Santa Cruz Biotechnology), or mouse monoclonal anti-ZO-1 antibody (Invitrogen; Carlsbad, CA) (all were diluted 1:50 in 1% BSA/PBS) at 37°C for 1 h. After three washes with PBS, the cells were incubated with Alexa Flour 488-conjugated anti-rat or anti-mouse IgG (Invitrogen) (1:500 in 1% BSA/PBS containing 1:1,000 Hoechst dye) at 37°C for 1 h. After mounting with slide, the cells were examined and imaged under a fluorescence microscope (Eclipse 80i) (Nikon). Quantitative intensity data were measured from at least 100 cells in ≥ 10 random HPFs per each biological sample using NIS-Elements D V.4.11 (Nikon).

Quantitative RT-PCR (qRT-PCR)
After 8-h incubation with or without COM crystals, culture medium was removed and the cells were washed with PBS. TRIzol reagent (Invitrogen) was added into each well for total RNA extraction. The solution was collected and RNA purification was performed using Direct-zol RNA MiniPrep (Zymo Research; Irvine, CA). cDNA was synthesized from an equal amount of purified total RNA via reverse transcription using SuperScript III reverse transcriptase (Thermo Fisher Scientific; Waltham, MA) and random primer. To assess mRNA expression levels of ARID1A, PTEN, VHL and TPX2, iTaq universal SYBR Green Supermix (Bio-Rad; Hercules, CA) was applied for qRT-PCR using specific primers as follows.
Summary of all primers used in this study.

Western blotting
After 24-h incubation with or without COM crystals, cellular proteins were solubilized by Laemmli's buffer, and protein concentrations were measured by Bradford's method. Western blotting was performed as reported previously [11,12]. Proteins were resolved by 12% SDS-PAGE with equal loading (50 µg/lane) and then transferred onto a nitrocellulose membrane. After blocking non-specific binders with 5% skim-milk/PBS for 1 h, the membrane was incubated overnight at 4°C with mouse monoclonal anti-ARID1A (Santa Cruz Biotechnology) or anti-GAPDH antibody (Santa Cruz Biotechnology) (1:1,000 in 1% skim milk/PBS). After washing with PBS three times, the membrane was incubated with rabbit anti-mouse IgG conjugated with horseradish peroxidase (1:20,000 in 1% skimmilk/PBS) (Sigma-Aldrich) at 25°C for 1 h. The immunoreactive protein bands were visualized by SuperSignal West Pico chemiluminescence substrate (Pierce Biotechnology, Inc.; Rockford, IL) and quantified by using ImageQuant TL software (GE Healthcare; Uppsala, Sweden).

Cell invasion assay
Cell invasion assay was performed as described previously [13,14]. Briefly, membrane insert of Transwell culture plate (5-µm pore size, 0.33-µm 2 surface area) (Corning Costar; Cambridge, MA) was pre-coated with Matrigel (BD Biosciences) at 37°C overnight. After 24-h incubation with or without COM crystals, the cells were detached and resuspended in non-supplemented MEM at a density of 1×10 6 cells/ml. Approximately 2×10 5 cells (200 μl) were seeded into upper chamber of each well, whereas the lower chamber was filled with 10% FBS-supplemented MEM. After 24-h incubation at 37°C in a humidified incubator with 5% CO2 for 24 h, the cells remained above membrane insert were removed, whereas those appeared under the membrane insert were fixed with 4% (w/v) paraformaldehyde/PBS for 15 min and stained with Hoechst dye (1:2,000 in PBS) at 25°C for 10 min. The invading cells were imaged under a fluorescence microscope (Eclipse 80i) (Nikon). Number of the invading cells was counted from at least 15 random low-power fields (LPFs) per each biological sample.

Cell-aggregate formation (hanging-drop) assay
Hanging drop assay was performed as described previously [13,14]. After 24-h incubation with or without COM crystals, the cells were detached and resuspend in 10% FBS-supplemented MEM at a density of 2.5×10 5 cells/ml. For each sample, a total of 10 drops of cell suspension (approximately 20 µl/drop containing 5×10 3 cells) was spotted on the inner side of upper lid, which was then placed on top of 100-mm culture dish containing 5-ml of 10% FBS-supplemented MEM. After 24-h incubation at 37°C in a humidified incubator with 5% CO2, the cells were harvested and pipetted up and down several times. Cell-aggregate formation was imaged under the Eclipse Ti-S inverted phase-contrast microscope (Nikon) and analyzed by using the NIS-Elements D software version 4.11 (Nikon). The cell-aggregate size was measured from at least 100 individual aggregates per each biological sample.